The invention concerns methods and compositions for the use of recombinagenic oligonucleobases in vivo for the correction of disease causing genetic defects and the prevention of disease by introducing genetic modifications into the genes that encode Apolipoprotein B (Apo B) and Apolipoprotein E (Apo E)
2.1 The Use of Chimeric Mutational Vectors to Effect Genetic Changes in Cultured Cells
The inclusion of a publication or patent application in this specification is not an admission that the publication or the invention, if any, of the application occurred prior to the present invention or resulted from the conception of a person other than the present inventors.
The published examples of recombinagenic oligonucleobases are termed Chimeric Mutational Vectors (CMV) or chimeraplasts because they contain both 2xe2x80x2-O-modified ribonucleotides and deoxyribonucleotides.
An oligonucleotide having complementary deoxyribonucleotides and ribonucleotides and containing a sequence homologous to a fragment of the bacteriophage M13mp19, was described in Kmiec, E. B., et al., November 1994, Mol. and Cell. Biol. 14, 7163-7172. The oligonucleotide had a single contiguous segment of ribonucleotides. Kmiec et al. showed that the oligonucleotide was a substrate for the REC2 homologous pairing enzyme from Ustilago maydis. 
Patent publication WO 95/15972, published Jun. 15, 1995, and counterpart U.S. Pat. No. 5,565,350 (the ""350 patent) described duplex CMV for the introduction of genetic changes in eukaryotic cells. Examples in a Ustilago maydis gene and in the murine ras gene were reported. The latter example was designed to introduce a transforming mutation into the ras gene so that the successful mutation of the ras gene in NIH 3T3 cells would cause the growth in soft agar of a colony of cells (xe2x80x9ctransformationxe2x80x9d). The ""350 patent reported that the maximum rate of transformation of NIH 3T3 was less than 0.1%, i.e., about 100 transformants per 106 cells exposed to the ras duplex CMV. In the Ustilago maydis system the rate of transformants was about 600 per 106. A chimeric vector designed to introduce a mutation into a human bcl-2 gene was described in Kmiec, E. B., February 1996, Seminars in Oncology 23, 188.
A duplex CMV designed to repair the mutation in codon 12 of K-ras was described in Kmiec, E. B., December 1995, Advanced Drug Delivery Reviews 17, 333-40. The duplex CMV was tested in Capan 2, a cell line derived from a human pancreatic adenocarcinoma, using LIPOFECTIN(trademark) to introduce the duplex CMV into the Capan 2 cells. Twenty four hours after the duplex CMV was introduced, the cells were harvested and genomic DNA was extracted; a fragment containing codon 12 of K-ras was amplified by PCR and the rate of conversion estimated by hybridization with allele specific probes. The rate of repair was reported to be approximately 18%.
A duplex CMV designed to repair a mutation in the gene encoding liver/bone/kidney type alkaline phosphatase was reported in Yoon, K., et al., March 1996, Proc. Natl. Acad. Sci. 93, 2071. The alkaline phosphatase gene was transiently introduced into CHO cells by a plasmid. Six hours later the duplex CMV was introduced. The plasmid was recovered at 24 hours after introduction of the duplex CMV and analyzed. The results showed that approximately 30 to 38% of the alkaline phosphatase genes were repaired by the duplex CMV.
WO 97/41411 and counterpart U.S. Pat. No. 5,760,012 to E. B. Kmiec, A. Cole-Strauss and K. Yoon, and the publication Cole-Strauss, A., et al., September 1996, SCIENCE 273, 1386 disclose duplex CMV that are used in the treatment of genetic diseases of hematopoietic cells, e.g., Sickle Cell Disease, Thalassemia and Gaucher Disease. U.S. Pat. No. 5,731,181 to E. B. Kmiec describes duplex CMV having non-natural nucleotides for use in specific, site-directed mutagenesis. The duplex CMV described in the applications and certain of the publications of Kmiec and his colleagues contain a central segment of DNA:DNA homoduplex and flanking segments of RNA:DNA hybrid-duplex or 2xe2x80x2-OMe-RNA:DNA hybrid-duplex.
The work of Kmiec and his colleagues concerned cells that are mitotically active, i.e., proliferating cells, at the time they are exposed to CMV. Kmiec and colleagues used a CMV/liposomal macromolecular carrier complex in which the CMV were mixed with a pre-formed liposome or lipid vesicle. In such a complex the CMV are believed to adhere to the surface of the liposome.
Kren et al., June 1997, Hepatology 25, 1462-1468, reported the successful use of a CMV in non-replicating, primary tissue-cultured rat hepatocytes to mutate the coagulation factor IX gene. Kren et al., March 1998, Nature Medicine 4, 285 reported the use of a CMV in vivo to introduce a genetic defect in the same gene.
2.2 The Use of a Polyethylenimine Macromolecular Carrier for In Vivo and In Vitro Transfection
Branched chain polyethylenimine has been used as a carrier to introduce nucleic acids into eukaryotic cells both in vivo and in vitro. Boussif, O., et al., 1995, Proc. Natl. Acad. Sci. 92, 7297; Abdallah, B. et al., 1996, Human Gene Therapy 7, 1947. Boletta, A., et al., 1997, 8, 1243-1251. The in vitro use of galactosylated polyethylenimine to introduce DNA into cultured HepG2 hepatocarcinoma cell lines is reported by Zanta, et al., Oct. 1, 1997, Bioconjugate Chemistry 8, 839-844. The coupling of a protein ligand, transferrin, to polyethylenimine and its use to introduce a test gene into cultured cells by use of the transferrin receptor is described in Kircheis, R., et al., 1997, Gene Therapy 4, 409-4-18. Branched chain polyethylenimines contain secondary and tertiary amino groups having a broad range of pK""s and, consequently these polyethylenimines have a substantial buffering capacity at a pH where polylysine has little or no capacity, i.e., less than about 8. Tang, M. K., and Szoka, F. C., 1997, Gene Therapy 4, 823-832. The use of branched chain polyalkanylimines, including polyethylenimine as carriers for the introduction of nucleic acids into cells is described in WO 96/02655 to J-P. Behr et al.
The successful in vivo and in vitro use of linear polyethylenimine to transfect a gene is reported by Ferrari, S., et al., 1997, Gene Therapy 4, 1100-1106. Compositions comprising a linear polyalkanylimine and a nucleic acid as disclosed in patent publication WO 93/20090 to S. Stein et al.
2.3 The Use of a Liposomal Carrier for In Vivo Transfection
The use of liposomes or lipid vesicles to introduce DNA encoding a foreign protein into cells has been described. The most frequently used techniques adhere the DNA to the surface of a positively charged liposome, rather than encapsulating the DNA, although encapsulated DNA techniques were known. U.S. Pat. Nos. 4,235,871 and 4,394,448 are relevant. The field is reviewed by Smith, J. G., et al., 1993, Biochim. Biophy. Acta 1154, 327-340 and Staubinger, R. M., et al., 1987, Methods in Enzymology 185, 512. The use of DOTAP, a cationic lipid in a liposome to transfect hepatic cells in vivo is described in Fabrega, A. J., et al., 1996, Transplantation 62, 1866-1871. The use of cationic lipid-containing liposomes to transfect a variety of cells of adult mice is described in Zhu, N., et al., 1993, Science 261, 209. The use of phosphatidylserine containing lipids to form DNA encapsulating liposomes for transfection is described in Fraley, R., et al., 1981, Biochemistry 20, 6978-87.
2.4 The Use of the Asialoglycoprotein Receptor for Hepatoceelular Specific Transfection
U.S. Pat. Nos. 5,166,320 and 5,635,383 disclose the transfection of hepatocytes by forming a complex of a DNA, a polycationic macromolecular carrier and a ligand for the asialoglycoprotein receptor. In one embodiment, the macromolecular carrier was polylysine. The use of a lactosylcerebroside containing liposome to transfect a hepatocyte in vivo is described by Nandi, P. K., et al., 1986, J. Biol. Chem. 261, 16722-16722. The use of asialofetuin-labeled liposomes to transfect liver cells with a reporter plasmid is described in Hara et al., 1995, Gene Therapy 2, 764-788. The use of galactosylated poyethyleneimine to transfect cultured hepatocytes is described in Zanta M-A., et al. abst. pub. Oct. 1, 1997, Bioconjugate Chem., 8, 839-844.
2.5 Apo B100, Apo B48 and the Reduction of Serum LDL
Hepatic and Intestinal Lipoprotein Secretion: Both the liver and the intestines make and export lipoproteins for the transport of lipids. The lipoproteins are termed very low density lipoproteins (VLDL) and chylomicrons, respectively. VLDL and chylomicrons differ in size and in their major protein components. The major protein of VLDL is Apo B100, consisting of 4536 amino acids; the major protein of chylomicrons is Apo B48, which consists of the N-terminal 2152 amino acids of Apo B100. Apo B48 and Apo B100 are encoded by a single gene, the transcript of which is modified at nucleotide 6666 (codon 2179) by a sequence specific cytidine deaminase, termed apolipoprotein B mRNA editing enzyme (APOBE). The action of this enzyme converts a C to U and results in a stop codon.
Both VLDL, which contain Apo B100, and chylomicrons, which contain Apo B48 transport triglycerides in the vascular system to a delivery site. However, after triglyceride hydrolysis and delivery VLDL are transformed into LDL, while chylomicrons are not. High levels of circulating LDL per se and a high LDL:HDL ratio increase the risk of arterial atherosclerosis. Hence, it has been suggested that increasing the ratio of Apo B48 to Apo B100 would have a beneficial effect.
In many species of mammals, e.g., rats and mice, a high percentage of the lipid secretions of both liver and intestine contain Apo B48. Such species have markedly lower ratios of LDL:HDL. Greve J., et al., 1995, Proc. Zool. Soc., Calcutta, 47, 93-100. In others, such as humans and rabbits, hepatocytes lack APOBE and the hepatocytes consequently produce only VLDL.
One strategy to reduce the atherosclerosis in humans has been to introduce the gene for the catalytic component of the apolipoprotein B editing enzyme (APOBEC-1) under the control of a constitutive promoter to convert Apo B100 transcripts into Apo B48 transcripts. The transient expression of APOBEC-1 in the hepatocytes of normal and genetically hyperlipidemic Watanabe rabbit does cause a transient reduction in the levels of LDL. Greeve, J., et al., 1996, J. Lipid Res. 37, 2001-17. However, the uncontrolled production of APOBEC-1 is mutagenic and may cause hepatocellular hyperplasia and hepatocellular carcinoma. Yamanaka, S., et al., 1995, Proc. Natl. Acad. Sci. 92, 8483-8487.
Individuals who are homozygous or mixed heterozygotes for genes encoding truncated Apo B100 have been observed. Malloy et al., 1981, J. Clin. Invest. 67, 1441; Hardman, D. A., et al., 1991, J. Clin. Invest. 88, 1722. These individuals have low or absent LDL. For example, deletion of nucleotides 5391-5394 results in a frame shift mutation and a shortened Apo B (B37). These patients are most often asymptomatic. Steinberg, D., et al., 1979, J. Clin. Invest. 64, 292; Young, S. G., et al., 1988, Science 241, 591; Young, S. G., 1987, J. Clin. Invest. 79, 1831. Reviewed Linton, M. F., 1993, J. Lipid. Res. 34, 521; Kane, J. P. and Havel, R. J., 1995, Chapt. 57, The Metabolic Basis of Inherited Disease, ed. Scriver et al. (McGraw Hill, New York). Similarly, as many as 1 in every 3,000 persons has a serum cholesterol level of 100 mg/dl or less because the individual is heterozygous for a truncated Apo B gene. Ibid., p. 1866.
Truncations that result in an Apo B that are shorter than Apo B 31 do not circulate. Truncated Apo B 86, 87 and 90 have been observed. Apo B 86 and Apo B 87, are not associated with LDL while Apo B 90 is. Each mutation is associated with hypobetalipoproteinemia. Linton, M. L., et al., 1990, Clin. Res. 38, 286A (abstr.); Tennyson, G. E., et al., 1990, Clin. Res. 38, 482A (abstr.); Kruhl, E. S., et al., 1989, Arteriosclerosis 9, 856.
2.6 Apo E Polymorphism and Type III Hyperlipidemia
Apolipoprotein E is the major ligand for the LDL receptor for lipoproteins that contain Apo B48. There are three allelic forms of human Apo E that differ from each other by one or two amino acids: Apo E2 (Cys112 Cys158); Apo E3 (Cys112 Arg158); and Apo E4 (Arg112 Arg158). There is considerable geographical variation in the prevalences of the alleles. Excluding Africa, E2 ranges between 4% and 12%, E3 between 70% and 85% and E4 between 7.5 and 25%. In the Sudan, the prevalences are 8.1%, 61.9% and 29.10%, respectively. Mahley, R. W. and Rall, S. C., Jr., 1995, Chapt. 61, The Metabolic Basis of Inherited Disease, ed. Scriver et al. (McGraw Hill, New York). Thus approximately 1% of the North American and European population are Apo E 2/2 homozygotes. Of these homozygotes approximately between 2% and 10% display type III hyperlipidemia. Paradoxically, however, Apo E 2/2 homozygotes that have not developed overt Type III hyperlipidemia display lower than average LDL associated cholesterol. Davignon, J., 1988, Arteriosclerosis 8, 1.
The E4 allele is also associated with increased incidence of a major disease, Alzheimer""s Disease, and with increased risk of coronary artery disease. Roses, A. D., 1996, Ann. NY Acad. Sci. 802, 50-57; Okumoto, K., and Fujiki, Y., 1997, Nature Genetics 17, 263; Kuusi, T., et al., 1989, Arteriosclerosis 9, 237. A polymorphism in the region 491 nt 5xe2x80x2 to the transcription start site of the Apo E gene is also and independently associated with increased risk of Alzheimer""s disease. Individuals homozygous for the xe2x88x92491-A genotype have an increased risk of Alzheimer""s, while individuals homozygous or heterozygous for the xe2x88x92491 T genotype have no increased risk. Bullido, M. J., 1998, et al., Nature Genetics 18, 69-71.
The E2 allele in most individuals is associated with the lowest levels of serum cholesterol and LDL. However, about 5% of E2/E2 homozygous persons who are subject to environmental or genetic stress develop type III hyperlipidemia. The most common stressors are hypothyroidism, untreated diabetes mellitus, alcoholism and marked weight gain. Removal of the stressor usually results in control of the hyperlipidemia. Rare patients with type III hyperlipidemia have mutant Apo I genes. Mahley and Rail, ibid. Table 61-5.
The present invention concerns methods of treatment and/or prophylaxis which consists of the introduction of specific genetic alterations in genes of a subject individual. In one embodiment, the specific genetic alteration blocks the synthesis of Apo B100 and thereby reduces the level of LDL cholesterol. In an alternative embodiment, the specific alteration converts an Apo E4 allele to an Apo E3 or Apo E2 allele, which is associated with decreased risk of atherosclerosis and Alzheimer""s Disease. In further alternative embodiments, the invention concerns the correction of inherited genetic defects in the genes of hepatocytes of individuals having a disease caused by such defects.
The invention can be practiced using any oligonucleotide or analog or derivative thereof, now known or hereafter developed, that can cause specific genetic alterations in the genome of the hepatocytes of the subject individual (hereafter a xe2x80x9crecombinagenic oligonucleobasexe2x80x9d), for example a chimeric mutational vector (CMV) as, for example, described in U.S. Pat. No. 5,565,350, No. 5,731,181, and No. 5,760,012. Alternatively, the recombinagenic oligonucleobase can be a heteroduplex mutational vector or a non-chimeric mutational vector as described in U.S. Pat. Nos. 6,004,804 and 6,010,907, each of which is hereby incorporated by reference.
In a preferred embodiment the recombinagenic oligonucleobase is complexed with a macromolecular carrier to which is attached a specific ligand. The ligand is selected to bind to a cell-surface receptor that is internalized into hepatocytes through clathrin-coated pits into endosomes. The cell surface receptors that bind such ligands are termed herein xe2x80x9cclathrin-coated pit receptorsxe2x80x9d. Examples of hepatic clathrin-coated pit receptors include the low density lipoprotein (LDL) receptor and the asialoglycoprotein receptor.
In specific embodiments the macromolecular carrier can be 1) an aqueous-cored lipid vesicle of between 25 nm and 400 nm diameter, wherein the aqueous core contains the CMV; 2) a lipid nanosphere of between 25 nm and 400 nm diameter, having a lipid core, wherein the lipid core contains a lipophilic salt of the CMV; or 3) a polycationic salt of the CMV. Examples of polycations for such salts include polyethylenimine, polylysine and histone H1. In one embodiment the polycation is a linear polyethylenimine (PEI) salt having a mass average molecular weight greater than 500 daltons and less than 1.3 Md. Alternatively the polycation can be a branched-chain polyethylenimine.